Multiplexed multiphoton fluorescence lifetime microscopy: Real time 3D imaging of protein-protein interactions by FRET

多重多光子荧光寿命显微镜：通过 FRET 对蛋白质-蛋白质相互作用进行实时 3D 成像

• 批准号: BB/I022074/1
• 负责人: Simon Ameer-Beg
• 金额: $ 60.44万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI022074%2F1
• 关键词: Multiplexed; multiphoton; fluorescence; lifetime; microscopy

From the earliest invention of the camera, humans have been seeking to observe processes that are too fast or too complicated for the human eye and brain to determine. The first time-laspe images of a running horse allowing us to understand its motion, the moment a bullet ripped through an apple - images, freezing a moment in time so that we can examine minute details. In cellular biology our understanding of cellular function continues to evolve as we observe complex dynamic processes played out under a microscope, captured by a camera at high speed and slowly revealing its hidden intricacy. As biologists ask every more complex questions, so we must develop more sophisticated tools to rationalise the complex data that we observe. Our current understanding of protein interaction in cells is informed, principally, through the use of microscopical tools to delineate localisation and compartmentalisation of signalling events within cellular organelles such as mitochondria. Further insight can be gained regarding protein association utilising the so called Förster resonance energy transfer (FRET) technique. FRET acts as a molecular ruler, enabling us to measure the relative separation between proteins or protein-domains on the nanometer length scale. Our work has focused on the determination of protein-protein interactions by FRET and high-resolution fluorescent lifetime imaging (FLIM). Unfortunately, these advanced techniques are relatively slow in capturing cellular events so that our desire to observe real-time the processes involved in, for example cell migration (directed motion often under the action of chemical gradient) are stymied. With this project, we seek to significantly speed up the acquisition of protein-interaction data to allows us to observe cellular signalling as it happens. This can be achieved through multiplexing of our excitation and detection channels to such an extent that we envisage a 1000-fold improvement in imaging with no loss of spatial resolution in the image. This work represents the state-of-the-art in functional imaging with the opportunity to observe complex cellular events in unpreceded detail: capturing an image of a T-cell as it surveys a cancer cell, forming dynamic 3-dimensional contacts and observing the protein signalling events that drive these processes; observing the moment a cell responds to a chemical stimulus at the level of single-proteins. The technology we will develop will drammatically improve our understanding of dynamic events within cells offering insight into drug interactions in diverse applications throughout the lifesciences.

从最早发明相机开始，人类就一直在寻求观察那些太快或太复杂的过程，而人眼和大脑无法确定。第一个时间-一匹奔跑的马的图像让我们了解它的运动，一颗子弹穿过苹果的瞬间-图像，冻结了一个时刻，以便我们可以检查微小的细节。在细胞生物学中，我们对细胞功能的理解不断发展，因为我们在显微镜下观察复杂的动态过程，由相机高速捕捉并慢慢揭示其隐藏的复杂性。随着生物学家提出越来越复杂的问题，我们必须开发更复杂的工具来合理化我们观察到的复杂数据。我们目前对细胞中蛋白质相互作用的理解主要是通过使用显微镜工具来描绘细胞器（如线粒体）内信号事件的定位和区室化。利用所谓的Förster共振能量转移（FRET）技术可以获得关于蛋白质缔合的进一步见解。FRET作为一个分子标尺，使我们能够测量蛋白质或蛋白质结构域之间的相对分离的纳米长度尺度。我们的工作集中在确定蛋白质-蛋白质相互作用的FRET和高分辨率荧光寿命成像（FLIM）。不幸的是，这些先进的技术在捕获细胞事件方面相对较慢，因此我们实时观察例如细胞迁移（通常在化学梯度作用下的定向运动）中所涉及的过程的愿望受到阻碍。通过这个项目，我们寻求显着加快蛋白质相互作用数据的获取，使我们能够观察细胞信号传导。这可以通过我们的激发和检测通道的多路复用来实现，达到这样的程度，即我们设想在图像中没有空间分辨率损失的情况下成像改善1000倍。这项工作代表了功能成像领域的最新技术，有机会以前所未有的细节观察复杂的细胞事件：在T细胞调查癌细胞时捕获T细胞的图像，形成动态三维接触并观察驱动这些过程的蛋白质信号传导事件;观察细胞在单一蛋白质水平上对化学刺激做出反应的时刻。我们将开发的技术将极大地提高我们对细胞内动态事件的理解，从而深入了解整个生命科学中各种应用中的药物相互作用。

项目成果

A 256 × 8 SPAD line sensor for time resolved fluorescence and raman sensing

256

• DOI: 10.1109/esscirc.2014.6942042
• 发表时间: 2014
• 作者: Krstajic N

C2c: turning cancer into chronic disease.

C2c：将癌症变成慢性病。

• DOI: 10.1186/gm555
• 发表时间: 2014
• 期刊: Genome medicine
• 影响因子: 12.3
• 作者: Beck S;Ng T
• 通讯作者: Ng T

Multifocal multiphoton microscopy with adaptive optical correction

• DOI: 10.1117/12.2000188
• 发表时间: 2013-02
• 作者: Simao Coelho;S. Poland;N. Krstajić;D. Li;J. Monypenny;R. Walker;D. Tyndall;T. Ng;R. Henderson-R.-Hend

Improving TCSPC data acquisition from CMOS SPAD arrays

改进 CMOS SPAD 阵列的 TCSPC 数据采集

• DOI: 10.1117/12.2032807
• 发表时间: 2013
• 作者: Krstajic N

The application of local hypobaric pressure - A novel means to enhance macromolecule entry into the skin.

• DOI: 10.1016/j.jconrel.2016.01.052
• 发表时间: 2016-03-28
• 期刊: Journal of controlled release : official journal of the Controlled Release Society
• 作者: Inacio R;Poland S;Cai XJ;Cleary SJ;Ameer-Beg S;Keeble J;Jones SA
• 通讯作者: Jones SA